Induction of primary NY-ESO-1 immunity: CD8 T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1 cancers Elke Ja ¨ ger* † , Sacha Gnjatic ‡ , Yasuhiro Nagata ‡ , Elisabeth Stockert ‡ , Dirk Ja ¨ ger*, Julia Karbach*, Antje Neumann*, Julia Rieckenberg*, Yao-Tseng Chen ‡§ , Gerd Ritter ‡ , Eric Hoffman ‡ , Michael Arand ¶ , Lloyd J. Old ‡ , and Alexander Knuth* *II. Medizinische Klinik, Ha ¨ matologie–Onkologie, Krankenhaus Nordwest, 60488 Frankfurt, Germany; ‡ Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan–Kettering Cancer Center, New York, NY 10021; § Cornell University Medical College, New York, NY 10021; and ¶ Institut fu ¨ r Toxikologie, Johannes Gutenberg Universita ¨ t Mainz, 55101 Mainz, Germany Contributed by Lloyd J. Old, August 29, 2000 Cancer–testis antigen NY-ESO-1 is one of the most immunogenic tumor antigens defined to date. Spontaneous humoral and CD8 T-cell responses to NY-ESO-1 are detected in 40 –50% of patients with advanced NY-ESO-1-expressing tumors. A clinical trial was initiated to study the immunological effects of intradermal vacci- nation with 3 HLA-A2-binding NY-ESO-1 peptides in 12 patients with metastatic NY-ESO-1-expressing cancers. Seven patients were NY-ESO-1 serum antibody negative, and five patients were NY-ESO-1 serum antibody positive at the outset of the study. Primary peptide-specific CD8 T-cell reactions and delayed-type hypersensitivity responses were generated in four of seven NY- ESO-1 antibody-negative patients. Induction of a specific CD8 T-cell response to NY-ESO-1 in immunized antibody-negative pa- tients was associated with disease stabilization and objective regression of single metastases. NY-ESO-1 antibody-positive pa- tients did not develop significant changes in baseline NY-ESO-1- specific T-cell reactivity. However, stabilization of disease and regression of individual metastases were observed in three of five immunized patients. These results demonstrate that primary NY- ESO-1-specific CD8 T-cell responses can be induced by intrader- mal immunization with NY-ESO-1 peptides, and that immunization with NY-ESO-1 may have the potential to alter the natural course of NY-ESO-1-expressing tumors. A nalysis of spontaneous immune responses against cancer in humans has led to the identification of a large number of tumor antigens (1). The majority of these antigens can be classified into one of the following categories according to their expression pattern, function, or origin: cancer–testis (CT) anti- gens, e.g., MAGE (2, 3) and NY-ESO-1 (4), which are aberrantly expressed in tumor cells but that, with the exception of germ cells, are silent in normal cells; differentiation antigens of the melanocyte lineage, e.g., Melan AMART-1 (5, 6), tyrosinase (7), and gp100 (8, 9); mutational antigens, e.g., MUM-1 (10), p53 (11, 12), and CDK4 (13); overexpressed ‘‘self’’ antigens, e.g., HER2neu (14) and p53 (12); and viral antigens, e.g., HPV (15) and EBV (16). Spontaneous immune responses elicited by these antigens are either predominantly cellular, e.g., tyrosinase (17, 18) and Melan AMART-1 (9, 19), or are associated with a strong humoral immune component, e.g., NY-ESO-1 (20) and p53 (12). NY-ESO-1 is a highly immunogenic CT antigen, inducing simultaneous cellular and humoral immune responses in a high percentage of patients with advanced NY-ESO-1-expressing tumors (20, 21). Detectable NY-ESO-1 serum antibody depends on the presence of NY-ESO-1-expressing tumor, and antibody titers correlate with the clinical development of disease (20, 22). NY-ESO-1-specific CD8+ T-cell responses were detected in more than 90% of NY-ESO-1 antibody-positive patients, whereas NY-ESO-1 antibody-negative patients showed no de- tectable NY-ESO-1-specific T-cell reactivity (23). The present study was initiated to evaluate the effects of active immunization with NY-ESO-1 peptides in NY-ESO-1 antibody- negative and -positive patients. Three naturally processed NY-ESO-1 peptides presented by HLA-A2 were used for intra- dermal immunization, first alone and then in combination with granulocyte–macrophage colony-stimulating factor (GM-CSF) as a systemic adjuvant. The following parameters were moni- tored in this trial: (i) peptide-specific CD8+ T-cell responses; (ii) delayed-type hypersensitivity (DTH) reactivity; (iii) NY-ESO- 1-specific antibody responses; and (iv) disease status. Methods Immunization Protocol. Twelve HLA-A2+ patients with progress- ing NY-ESO-1-expressing metastatic tumors of different types and meeting predefined entry criteria were selected for immu- nization in the LUD97-008 protocol sponsored by the Ludwig Institute for Cancer Research. Immunizations were performed with three HLA-A2-binding NY-ESO-1 peptides derived from NY-ESO-1 and initially identified by the T-cell line NW38-IVS-1 (21). The NY-ESO-1 peptide sequences were: p157–167 (SLLM- WITQCFL), p157–165 (SLLMWITQC), and p155–163 (QLSLLMWIT). The HLA-A2-presented inf luenza matrix pep- tide p58–66 (GILGFVFTL) was used as a positive control for immune responses in vitro and in vivo. Peptides (90% purity) were manufactured according to good manufactorial practice guidelines (Multiple Peptide Systems, San Diego) and solubi- lized in 100% DMSO. Intradermal injection of the 100% DMSOpeptide solution caused an immediate nonspecific skin reaction. For this reason, the peptide solution was diluted with PBS to a final concentration of 33% DMSO, and this concen- tration of DMSO caused no local toxic skin reaction. For immunization, the total dose of 100 g of each peptide solubi- lized in a final volume of 0.9 ml 33% DMSO was divided in 3 portions of 0.3 ml each and was injected intradermally at separate sites once weekly for 4 weeks. Patients with no evidence of disease progression on day 50 received further immunizations by using the peptides at the same dose and schedule, combined Abbreviations: CT, cancer-testis; DTH, delayed-type hypersensitivity; ELISPOT, enzyme- linked immunospot; GM-CSF, granulocyte–macrophage colony-stimulating factor. † To whom reprint requests should be addressed. E-mail: EJ200161@AOL.com. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073pnas.220413497. Article and publication date are at www.pnas.orgcgidoi10.1073pnas.220413497 12198 –12203 | PNAS | October 24, 2000 | vol. 97 | no. 22